Expression systems for the production of recombinant polypeptides are well-known and reported in the state of the art literature. For the production of polypeptides used in pharmaceutical applications mammalian cells such as CHO cells, BHK cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, PER.C6® cells, and the like are employed. The nucleic acid encoding the polypeptide is introduced into the cell e.g. in a plasmid, such as, for example, an expression plasmid. The essential elements of an expression plasmid are a prokaryotic plasmid propagation unit, e.g. for Escherichia coli comprising an origin of replication and a selection marker, a eukaryotic selection marker, and one or more expression cassettes for the expression of the nucleic acid(s) of interest each of them comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. For transient expression in mammalian cells a mammalian origin of replication, such as the SV40 On or OriP, may be included. As a promoter a constitutive or inducible promoter can be selected. For optimized transcription a Kozak sequence may be included in the 5′ untranslated region. For mRNA processing a polyadenylation signal may be included as well.
Proteins and especially immunoglobulins play an important role in today's medical portfolio. For human application every pharmaceutical substance has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans substances, which would cause severe harm, have to be removed especially.
The splicing of mRNA is regulated by the occurrence of a splice donor site in combination with a splice acceptor site, which are located at the 5′ end and 3′ end of an intron, respectively. According to Watson et al. (Watson et al. (Eds), Recombinant DNA: A Short course, Scientific American Books, distributed by W.H. Freeman and Company, New York, N.Y., USA (1983)) are the consensus sequence of the 5′ splice donor site ag|gtragt (exon|intron) and of the 3′splice acceptor site (y)nNcag|g (intron|exon) (r=purine base; y=pyrimidine base; n=integer; N=any natural base).
In 1980 first articles dealing with the origin of secreted and membrane bound forms of immunoglobulins have been published. The formation of the secreted (sIg) and the membrane bound (mIg) isoform results from alternative splicing of the heavy chain pre-mRNA. In the mIg isoform a splice donor site in the exon encoding the C-terminal domain of the secreted form (i.e. the CH3 or CH4 domain, respectively) and a splice acceptor site located at a distance downstream thereof are used to link the constant region with the downstream exons encoding the transmembrane domain.
A method to prepare synthetic nucleic acid molecules having reduced inappropriate or unintended transcriptional characteristics when expressed in a particular host cell is reported in WO 2002/016944. In WO 2006/042158 are reported nucleic acid molecules modified to enhance recombinant protein expression and/or reduce or eliminate mis-spliced and/or intron read through by products.
Therefore there exists a need for a recombinant production method for immunoglobulins with reduced by products.